Exosomal miRNA‐21‐5p and miRNA‐21‐3p as key biomarkers of myocardial infarction

Abstract Objective Coronary artery disease (CAD) is a debilitating condition that can lead to myocardial infarction (MI). Exosomal miRNAs (exo‐miRNA) can be diagnostic biomarkers for detecting MI. Here, we conduct a study to evaluate the efficacy of exo‐miRNA‐21‐5p/3p for early detection of MI. Methods A total of 135 CAD patients and 150 healthy subjects participated in this study. Additionally, we randomly divided 26 male Wistar rats (12 weeks old) into two groups: control and induced MI. Angiographic images were used to identify patients and healthy individuals of all genders. In the following, serum exosomes were obtained, and exo‐miRNA‐21‐5p/3p was measured by reverse‐transcriptase polymerase chain reaction. Results We observed an upregulation of exo‐miRNA‐21‐5p/3p in CAD patient and MI‐induced animal groups compared to controls. Analysis of the ROC curves defined 82% and 88% of the participants' exo‐miRNA‐21‐5p and exo‐miRNA‐21‐3p diagnostic power, respectively, which in the animal model was 92 and 82. Conclusion This study revealed that the mean expression levels of exo‐miRNA‐21‐5p/3p were significantly increased in CAD patients and animal models of induced MI. Also, these results are associated with the atherogenic lipid profile of CAD patients, which may play an important role in the progression of the disease. Therefore, they can be considered as novel biomarkers.


| INTRODUCTION
Coronary artery disease (CAD) occurs when coronary arteries narrow due to atherosclerosis or clots, causing insufficient oxygen supply, angina, and myocardial infarction (MI). 1,2A proper diagnostic technique for the detection of CAD patients will help physicians initiate treatment more efficiently. 3Diagnosis, follow-up, and treatment of CAD involve numerous diagnostic methods, but common tests like exercise electrocardiography, 4 coronary angiography, 5 and myocardial perfusion single-photon emission computed tomography (SPECT) are limited by low sensitivity, invasiveness, high cost, accessibility, and radiation exposure. 6,7Although traditional methods like serum levels of troponin (Tn) and creatine kinase-MB (CK-MB) can diagnose MI, these enzymes typically increase after cardiomyocyte apoptosis and MI. 8 Therefore, the existence of a novel diagnostic target for early detection of heart cell damage is crucial. 9tracellular vesicles (EV) or exosomes, with an average diameter of 30-100 nm, could be a new diagnostic method, containing genetic information like DNA, mRNA, and microRNA (miRNAs) that can transmit various signals between different cells. 10MiRNAs are small noncoding RNAs with several activities, such as cell proliferation, gene regulation, and pathophysiological progression. 11,12Exosomal miRNAs (exo-miRNAs), which are more stable than plasma miRNAs due to their protection from body fluid degradation, 13 have been linked to heart injuries in humans and animals. 14Tao Geng et al.
showed exo-miRNA-143 decreased in MI, which enhanced angiogenesis. 15Evidence indicates that some miRNAs significantly contribute to the progression of heart disease, including myocardial ischemia, while others enhance function. 16,17Impaired blood flow in CAD can alter miRNA expression, leading scientists to study miRNA-21 as a potential diagnostic biomarker due to its significant difference in expression in CAD patients compared to healthy subjects. 18The gene of miRNA-21, located on chromosome 17q23.2, is responsible for the precursor miRNA-21's transcription and processing.This process creates two mature strands: the guide strand (miRNA- 21-5p)   and the passenger strand (miRNA-21-3p), which degrade due to lower stability. 19Mengwen Yan et al. revealed that miRNA-21-3p expression increases in heart failure patients and can control cardiac hypertrophic by inhibiting HDACs, which activate the Akt/Gsk3b pathway. 20Consistent with Mengwen Yan, WeiHuang showed that miRNA-21-5p inhibits cardiomyocyte apoptosis by restricting the PTEN/AKT pathway.In addition, Argonaute-miRNA-21 (agomiRNA-21) increased ejection fraction (EF) status and reduced the size of the infarct area in mice. 21Therefore, many studies have emphasized the therapeutic and diagnostic effects of miRNA-21.Zhou et al. suggest that miRNA-21 may play a potential therapeutic role.They showed that overexpression of miRNA-21 has a protective function by inhibiting the PTEN signaling pathway and apoptosis to prevent hypoxia-reperfusion (HR) and myocardial injury. 22This research aims to assess the diagnostic properties of exo-miRNA-21-5p/3p in CAD patients to distinguish between patients and healthy subjects.To determine exo-miRNA-21-5p/3p expression levels, we collected male Wistar rat serum from induced MI and control groups.This is the first study to consider exo-miRNA-21-5p and exo-miRNA-21-3p expression in CAD patients simultaneously, along with their biochemical factors.

| Human subjects
This study randomly included persons over 35 years of age: 135 patients with coronary artery disease, including subjects undergoing angiography at Ghaem Hospital, and 150 healthy participants.We classified people who underwent coronary angiography and had coronary stenosis >50% as coronary patients. 23Exclusion criteria for patients included: <50% stenosis, history of chronic systemic and inflammatory disease, cardiomyopathy, pregnant or lactating women, renal abnormalities, and immunosuppressive drugs such as azathioprine and tacrolimus.Also, healthy subjects were without symptoms, signs and history of angiography for coronary artery disease.In addition, Exclusion criteria for healthy group included: hypertension, vascular disease, dyslipidemia, diabetes mellitus, history of stroke and infarction, dyslipidemia and acute hypercholesterolemia family history, family history of coronary artery disease.

| Animal samples
According to the animal table, 26 male Wistar rats (weight 250-300 g and 12 weeks old) were equally divided into induction of MI and control groups.Each group was kept in its own cage, and standard conditions such as the proper temperature (25°C), humidity, light, sufficient water, and food were produced in the animal room of the Mashhad Faculty of Medicine for both groups.

| Ethics
Participants provided informed written consent.Also, Anonymity and confidentiality were maintained throughout the study.Furthermore, all experimental protocols were performed according to the guidelines approved by the ethics committee of Mashhad University of Medical Sciences (MUMS).

| The laboratory characteristics and clinical baseline of all subjects
As shown in Table 1, all CAD patients and healthy group have similar ages.Patients and the control group provided information on lowdensity lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), body mass index (BMI), total cholesterol (TC), systolic blood pressure (SBP), diastolic blood pressure (DBP), highsensitivity C-reactive protein (hs-CRP), and smoking.Total cholesterol, hs-CRP, LDL-C, and HDL-C were in the patient's record before angiography.The patient's blood pressure was monitored upon entrance to the angiography department according to World Health Organization guidelines. 24Cardiologists assessed the angiographic data.Positive findings (stenosis above 50%) were considered a diagnostic criterion of vascular stenosis.To eliminate observer bias, only one cardiologist examined the results in this study.

| Animal MI induction using isoprenaline hydrochloride
In this study, the induction procedure for MI using isoprenaline hydrochloride (ISO) was performed according to the methods of previous studies. 25,26First, all animals were weighed on an electronic scale.Next, 150 mg of ISO powder (Sigma-Aldrich) was dissolved in 2 mL of water for injection.150 mg/kg of this solution was then injected subcutaneously for 2 consecutive days.Immediately after the last injection, exosomes were extracted from serum samples, and exo-miRNA-21-5p expression levels were assessed by quantitative PCR (qPCR).We injected the healthy group with an equal volume of saline (pH: 7.2) for 2 days to equalize the injection stress between the MI rats and the control group.

| Blood sampling
Blood samples (5 mL) from human subjects were collected in ethylenediaminetetraacetic acid (EDTA) tubes from the antecubital vein in a sitting position.In addition, for animal samples, all animals were anesthetized with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (10 mg/kg), and 2 mL of blood was collected from the sinus membrane of rat eyes by inserting a capillary tube.
Finally, blood samples were immediately centrifuged at 2800 rpm for 15 min to separate serum and frozen at −80°C for future analysis.

| Exosome isolation
Serum samples were centrifuged at 2800 rpm for 12 min to remove sediment debris.We then obtained the serum exosomes using the exosome isolation kit (CIBBIOTECH) according to its method.Purified exosomes were stored at −80°C.The exosome size was assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

| DLS
The diameter of the exosome was determined by the DLS technique with a particle size analyzer (Vasco Instruments, Cordouan Technologies).In this study, phosphate-buffered saline (PBS) was used, and its refractive index was 1.33 with a viscosity of 0.891.We diluted 20 μL of purified exosomes in 100 μL of phosphate salt buffer (1:5).
This information is needed to analyze the results.

| TEM
For TEM analysis, 5 μL of exosome suspension was resuspended in PBS and then fixed with 1% glutaraldehyde.Ten microliters of the sample was placed on a carbon-coated grids at room temperature to dry.The grid was washed twice with sterile PBS for 5 min and finally stained with 1% uranyl acetate for 5 min.Finally, we observed the samples under TEM.

| RNA extraction and polyadenylation reaction
Total RNA was extracted from 1 mL of serum using the protocolbased RNX-PLUS kit (SINACLON) and according to the manufacturer's instructions.The concentration of RNA samples was measured by nanodrop at 260/280 nm (Thermo Scientific).Then, the polyadenylation reaction was carried out by BON-miRNA polyadenylation kit protocol (Bonbiotech).

| Complementary DNA (cDNA) synthesis
We synthesized the first strand of cDNA by using the BON-miRNA cDNA synthesis kit (Bon-Biotech), as described by the manufacturers.

| Relative quantification PCR
The SYBR Green real-time PCR reaction was used to evaluate the expression levels of exo-miRNA-21-5p and exo-miRNA-21-3p on the ABI STEP ONE real-time PCR system (Applied Biosystems).It was accomplished by according to the BON-miRNA QPCR kit (Bon-Biotech) protocols.

| Validation of serum-derived exosomes characterization using TEM and DLS
As shown in Figure 2, the structure and size distribution of serumderived exosomes were determined by TEM and DLS techniques.
TEM demonstrated that (Figure 2A) the integrity of the exosomes' membrane and spherical morphology were preserved during the separation process and the spherical structure was not disassembled.
Also, DLS measurements indicated the diameter of the serumderived exosomes was approximately 100 nm (Figure 2B).According to the accuracy of the above results, isolated exosomes could be used in further studies.

| Animal samples
ISO is a catecholamine and causes severe myocardial necrosis. 29,30The results showed in Figure 4

| Histopathological alteration in myocardium
MI causes cardiomyocyte cell damage and cell necrosis by leakage of lysosomal enzymes from damaged cells into the cytoplasm. 31The results of the necrosis study by light microscopy and using hematoxylin and eosin staining groups are as follows in the control group and MI (Figure 5).The myocardial tissue in the control group  The D'Agostino-Pearson, Shapiro-Wilk, and Kolmogorov-Smirnov normality tests showed that all data from both control and MI were normally distributed (p > 0.05).Next, we evaluated the expression levels of serum exo-miRNA-21-5p/3p in the MI and control groups (Figure 6).Also, the average fold changes in the exo-miRNA-21-5p and exo-miRNA-21-3p genes in the MI group compared with the control group were 11.4-and 6.38-fold (p < 0.05).

| DISCUSSION
CAD is one of the main causes of morbidity and mortality worldwide. 2rum troponin and CK-MB levels are the key MI diagnostic tests.
Although, cardiac troponin is used to diagnose MI because of its high sensitivity and specificity; however, its blood level increases only 3-4 h after cardiac injury.According to several reports, the presence of some interfering factors, such as rheumatoid factor and fibrin clots, SAHEBI ET AL.
| 7 of 10 leads to a false positive assay in troponin levels. 32These biomarkers cannot be used in early detection 33 ; thus, appropriate diagnostic methods are needed to more effectively treat and reduce mortality. 34ny researchers have studied miRNA in molecular and cellular functions, and disease processes in recent years. 35,36MiRNAs, also known as noncoding RNAs, play critical roles in apoptosis, cell proliferation, and angiogenesis. 37,38Cell damage and disease release a large number of miRNAs into the blood quickly.Therefore, they are valuable candidates for prognosis and diagnosis in many chronic diseases, including CAD. 39 Due to their small size and linking with carriers such as extracellular vesicles (EVs), circulating miRNAs are resistant to RNase activity.This stability makes them suitable for CAD diagnostic marker. 39Recently, miRNA-21 has been introduced as a potential biomarker for CAD and AMI diagnosis. 40Wang et al.
showed miRNA-21 could be involved directly and indirectly in the pathophysiological processes of atherosclerosis through apoptosis inhibition. 41,42Dong Yiheng discovered that miRNA-21 was upregulated in cardiac graft fibrosis.Also, this miRNA is overexpressed in patients with rejection of heart transplantation. 43Li Qiao and colleagues reported in 2019 that exosomes containing miRNA-21-5p help with heart repair by increasing angiogenesis and enhancing the expression level of vascular endothelial growth factor (VEGF) in patients with heart failure.Also, they showed exo-miRNA-21-5p is a mediator of the Akt pathway with the aim of cardiomyocyte survival. 44Furthermore, Zhang and coworkers illustrated in their research on rats with AMI that upregulation of miRNA-21 could improve cardiac function by reducing myocardial cell apoptosis. 45In agreement with Zhang JC's research, Liao showed exo-miRNA-21 had an antiapoptotic function and prevented the apoptosis of cardiomyocytes, causing the proliferation of endothelial cells by downregulating programmed cell death 4 (PDCD4). 46In this study, we compared the relationship between various demographic and biochemical parameters such as age, gender, BMI, LDL-C, hs-CRP, and alters in serum exosomal miRNA-21-5p/3p expression in patients with CAD in comparison with healthy subjects.We also evaluated the expression level of serum exo-miRNA-21-5p/3p in samples from rats that induced MI, as well as in the control group.
Our statistical analysis of the data indicates that exo-miRNA-21-5p/ 3p has a potential role in the diagnosis of coronary artery disease.
According to the results, risk factors like HDL-C, hs-CRP, and SBP are significantly related to high expression of exo-miRNA-21-5p/3p in the CAD group.Additionally, the association between smoking and exo-miRNA-21-5p/3p expression is considered a risk factor, but its impact is not significant.Some studies by Guo-fu Zhu and others in 2019 showed miRNAs related to inflammation (such as miRNA-21) are highly expressed, which is obviously positively correlated with the increase in the levels of inflammatory markers such as hs-CRP in coronary artery stenosis. 47Similarly, other research on patients with stable CAD has shown that elevating serum miRNA-21 may contribute to the development of inflammatory pathways in the pathogenesis of CAD or may be related to the effects of inflammatory diseases on atherosclerosis and acute coronary syndrome. 48 In confirmation of our hypothesis, Nana He et al. stated that exo-miRNA-21-5p could play a protective role for myocardial cells against apoptosis and could be used as a novel therapy for cardiovascular disease. 51Also, Ken Watanabe showed upregulation of the level of miRNA-21 expression occurs after angiotensin II (Ang II) infusion in mice, which reduces the expression level of PDCD4 compared to the control group. 52Accordingly, our reviews, along with other studies, suggest elevating the serum level of exo-miRNA-21-5p may be effective for the diagnosis of AMI, and this study considers this issue through ROC curve analysis.As a result, in rats, exo-miRNA-21-5p can predict AMI status with 92.3% sensitivity and 100% specificity.
Similar to our results, Feng Wang, based on the results of the ROC analysis in the cohort study, revealed that miRNA-21-5p could be a powerful diagnostic biomarker in AMI patients. 53In addition, Han Ding and colleagues illustrated in 2020 that miRNA-21-5p could be considered a clinically rapid diagnostic and prognostic biomarker with 89.7% sensitivity and 82.8% specificity for heart failure. 54At the end, despite considering most research information, our research has limitations: First, although the estimated population is small, the results seem to be consistent.Second, we have organized limited regional studies, and future investigations need to be conducted in
an increase in CK-MB and troponin I after ISO-induced stroke in the MI group compared with the control group.The mean serum CK-MB activity in the control and MI groups was 86 ± 1 and 222 ± 8 IU/L, respectively, indicating a significant difference (p < 0.0001).Furthermore, ROC curve analysis indicated that elevated CK-MB levels (>144.9IU/L) could identify the occurrence of stroke with 100% specificity and sensitivity.The mean troponin I concentration in the control group was 0.29 ng/mL, indicating a significant difference (p < 0.0001) compared with the MI group of 1.24 ng/mL.The ROC curve results can be used to diagnose stroke for troponin I at concentrations greater than 0.68 ng/mL with 100% specificity and sensitivity in rats.
was observed to be fascicles of muscle with longitudinal connections and without necrosis or inflammatory cell infiltration.In the MI group, superficial fibroid necrosis was observed along with transverse striations, muscle fiber rupture, inflammatory cell infiltration, and bleeding.F I G U R E 4 Comparison of creatine kinase-MB (A) and Troponin I (B) concentrations in control group and acute myocardial infarction.IU, international unit, ***p < 0.0001.F I G U R E 5 (A) Histopathological normal myocardial tissue with longitudinal connections and no necrosis and infiltration of inflammatory cells in control group.(B) The rupture of muscle fibers with loss of transverse striations in myocardial infarction group.F I G U R E 6 The relative expression levels of serum exosomal micoRNA-21-5p/3p.F I G U R E 7 Receiver-operating-characteristic curve of miRNA-21, (A) Diagnostic potential of miRNA-21-5p, area under the curve: 0.92 (0.79-1 at 95% confidence interval [CI]) with 100% specificity and 92.3% sensitivity, (B) Diagnostic potential of miRNA-21-3p, 0.82 (0.65-1 at 95% CI) with 85% specificity and 84% sensitivity.

4. 8 |
Expression of exo-miRNA-21-5p in MI and control groups multiple centers.Third, the lack of follow-up in the human models prevents us from exploring the possible role of exo-miRNA-21-5p/3p in CAD prognosis.Eventually, more patients from the general cardiac population will need to be screened for further study to clarify the relationship observed at the human molecular level.Exo-miRNA-21-3p overexpression positively correlates with hs-CRP and LDL-C levels in patients with coronary artery disease, while it negatively correlates with HDL-C.The correlation between exo-miRNA-21-5p and biochemical factors (HDL-C, LDL-C, and hs-CRP) is opposite to that between exo-miRNA-21-3p, but the results are not significant.Obviously, exo-miRNA-21-5p/3p play a key role in the lipid profile.It is necessary to determine the role of exo-miRNA-21 as a target for coronary artery disease diagnosis and treatment.Also, our study examined changes in the expression level of serum-derived exo-miRNA-21-5p after induction of MI in an animal model, and we confirmed that the expression of exo-miRNA-21-5p/3p increased in the MI group compared to the control.AUTHOR CONTRIBUTIONS Reza Sahebi: Writing-original draft; investigation; methodology; project administration; supervision; validation; conceptualization; funding acquisition; visualization.Fatemeh Gandomi: Writingreview and editing; data curation.Mitra shojaei: Writing-review and editing.Effat Farrokhi: Formal analysis; software; data curation; resources.
Table 2 lists the primers used in this study.Comparison of the baseline characteristics between different studied groups.
28te: Data are shown as mean ± SD or median (IQR); T-Student test and Mann-Whitney U test were used.Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive Protein; IQR, interquartile range; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; SD, standard deviation; TC, total cholesterol.*pValuesignificantlevel,<0.05.28A graph of relative expression levels plotted with GraphPad Prism version 8. Statistical significance was determined when the p < 0.05.4 | RESULTS4.1 | Human subjectsThis study included 135 patients with coronary artery disease with a mean age of 54 ± 10 years, as well as 150 healthy subjects defined as controls with a mean age of 53.1 ± 11.5 years.14.07% of the total population were smokers, and there was no significant difference between CAD and the control group.Clinical features, including HDL-C, LDL-C, TC, BMI, SBP, DBP, and hs-CRP, were collected in Table1.There was no significant difference (p > 0.05) in gender, age, LDL-C, TC, DBP, and BMI, between coronary patients and healthy subjects.The mean HDL-C level was significantly lower (34.27 ± 7.77 mg/dL) in the CAD group compared with the control group (56.15 ± 10.12 mg/dL).In addition, the t-test showed that the hs-CRP and SBP of the patients were significantly higher (p < 0.05).determine the regularity of the distribution of variables.Results indicated that all data from the CAD and control groups were not normally distributed (p < 0.05), and the Mann-Whitney U test was used.∆∆2Ct was used to assess the expression level of exo-miRNA-21-5p/3p in CAD and control groups.Figure1shows that the relative serum exo-miRNA-21-5p expression was significantly higher In line with previous reviews, the present study indicates exo-miRNA-21-3p has a significant positive correlation with hs-CRP levels in CAD patients.Compared with exo-miRNA-21-3p, exo-miRNA-21-5p has a negative correlation with hs-CRP levels, but this relationship is not significant.Hence, a significant increase in the expression level of exo-miRNA-21-5p/3p may be in response to CAD. Also, for the first time, the results of our investigation on animal-induced MI samples showed significant overexpression (p < 0.05) of exo-miRNA-21-5p/3p in serum samples of MI rats compared with the control group.This increasing level may promote a key pathway to reduce apoptosis and preserve cardiomyocyte cells.